Mass spectrometry (MS) has become a central tool in many fields of bioanalytical science, such as proteomics and metabolomics. The power of MS is steadily increasing with regard to sensitivity, mass accuracy, resolving power and precision of detection. With modern instruments, biological samples can be analyzed in a broad dynamic range (around 4 orders of magnitude) and mass-to-charge ratio (m/z) range (around 200 to 4,000) with high resolution (>100,000) and acquisition rates (>10 Hz). A limiting factor for direct mass analysis is the sequencing capability of MS. The specificity and the accuracy of analyte identification often suffer from the insufficient capacity of mass spectrometers to generate informative fragmentation pattern characteristics for the precursor ions. The efficiency of tandem MS (MS/MS) is particularly limited for large proteins. This is a major reason preventing wider application of top-down approaches, especially to protein-molecule complexes obtained by “native” MS.
The most commonly used method of fragmentation is referred to as Collision-Activated or Collision-Induced Dissociation (CAD/CID). In CAD/CID, accelerated precursor ions undergo multiple collisions with neutral gas molecules, resulting in gradual vibrational heating followed by the ultimate dissociation of the weakest bonds. Polypeptide precursor ions preferentially dissociate across backbone C—N bonds, yielding ‘b’ (N-terminal) and ‘y’ (C-terminal) fragments, respectively. An advantage of CAD is the relatively short time needed to generate abundant fragmentation (in the order of milliseconds) and easy technical implementation. One of the key limitations of CAD relevant to biological analyses is its poor sensitivity to the presence of Post-Translational Modifications (PTMs). Small PTMs, such as phosphorylation or sulfation functional groups, are often weakly bound to the polypeptide backbone and tend to be easily lost during activation, which prevents their observation in tandem MS. Besides that, the efficiency of sequencing based on CAD MS/MS commonly suffers from incomplete fragmentation along the peptide backbone. Finally, CAD is rather inefficient for large proteins because the energy supplied during the activation dissipates across the large number of vibrational modes. As a result, only a few peptide bonds fragment: those that receive vibrational heating sufficient for their dissociation.
Distinct from CAD/CID are Electron Capture/Transfer Dissociation (ECD/ETD) techniques, in which precursor ions receive an electron. Such techniques are described in U.S. Pat. No. 7,145,139 and U.S. Pat. No. 6,995,366. Electron addition to closed-shell molecular cations converts them to unstable radical cations of the “hydrogen-abundant” type. In addition, 4-7 eV of recombination energy is deposited, which adds a degree of vibrational heating. Electron transfer to multiply protonated polypeptides preferentially induces fast, perhaps even nonergodic, cleavage of N—Cα backbone bonds, yielding ‘c’ (N-terminal) and ‘z’ (C-terminal) types of fragments. In many cases, the high speed of the primary ECD process prevents significant redistribution of the recombination energy among vibrational modes prior to dissociation. As a result, loosely bound functional groups, such as found in protein-molecule complexes and in proteins with labile PTMs, can “survive” dissociation and be localized in the generated c and z fragments.
The sequence preferences in ETD/ECD and CAD/CID are complementary and the combination of their MS/MS data greatly facilitates spectral interpretation and reduces the rate of misidentifications in proteomics analyses. In high-resolution Fourier transform mass spectrometry (FTMS), the combined use of ECD and CAD has been demonstrated to improve the validity of the database search data by 20 to 100 times and to result in substantially higher number of identified proteins compared to CAD-only analysis. The cross-section of electron capture rapidly increases with the ionic state of precursor polypeptides, making it particularly suitable for the fragmentation of highly protonated species. For instance, electron capture cross section of cytochrome c+15 ions measured at typical ECD conditions exceeded the ion-neutral collision cross section by two orders of magnitude.
Alongside with ECD and ETD, a number of tandem MS techniques have been introduced that employ electron activation. In Electron Detachment Dissociation (EDD), deprotonated polypeptides are charge-reduced through the collisions with free electrons. This is described in Budnik, B. A.; Haselmann, K. F.; Zubarev, R. A. Chem. Phys. Lett. 2001, 342, 299. Reduced radical species dissociate along the Cα—C backbone giving rise to ‘x’ and ‘a’ fragments. In Metastable-Induced Dissociation (MIDI), electronically excited atoms of noble gas are used as electron donors to activate cationic polypeptides, resulting in fragmentation pattern similar to ECD/ETD. This is described in US-2005/258353 and Misharin, A. S.; Silivra, O. A.; Kjeldsen, F.; Zubarev, R. A. Rapid Commun. Mass Sp. 2005, 19, 2163, and Berkout, V. D., Anal. Chem. 2006, 78, 3055 for example. Alternatively, electron transfer to polypeptides can also be induced via high-energy collisions with alkali metal vapours.
The efficiency (determined as the ratio of the product ion abundances versus the precursor ion abundance) of ECD/ETD strongly depends on the charge state of precursor species. At low charge states, the efficiency of these techniques tends to be limited, especially for 2+ precursors, for which one of the fragments is by necessity neutral. The latter limitation may represent a serious problem for shotgun proteomics, in which the most of analyzed proteolytic peptides are doubly charged. Thus, increasing the charge state of precursor ions prior to fragmentation would enhance the efficiency of subsequent activation by electron transfer and enable the application of ECD/ETD to singly-charged precursors. Such a “supercharging” of analyte cations can be achieved via Electron Ionization Dissociation (EID) of trapped ions, such as described in GB-2 405 526, U.S. Pat. No. 6,800,851 and Fung, Y. M. E.; Adams, C. M.; Zubarev, R. A. J. Am. Chem. Soc. 2009, 131, 9977. However, the difficulty of dealing with electron beams entering a radiofrequency multipole may have prevented such a method from being widely applied.
An improved method of ion fragmentation is therefore desirable, especially one suited to MS/MS analysis of biological samples.